Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities

Abstract

TMPRSS6 (matriptase‐2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA‐seq data and RT‐PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6‐1) and 3 are mostly expressed in human testis while TMPRSS6‐2 and TMPRSS6‐4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6‐1 undergoes internalization. Moreover, truncated TMPRSS6‐3 or catalytically altered TMPRSS6‐4 interact with HJV and prevent its cleavage by TMPRSS6‐2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.

Functional diversity of TMPRSS6 isoforms and variants expressed in hepatocellular carcinoma cell lines

“…cDNA encoding TMPRSS6-2 WT, TMPRSS6-1 S762A and HJV were obtained and cloned as previously described 23 , 24 . TMPRSS6-2 constructs V736A, V795I, G603R and S762A were obtained using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA).…”

“…Laser excitation was performed at 405 nm (50 mW Violet diode laser) and 488 nm (40 mW Blue Argon Laser). Images were pseudocoloured according to their original fluorochrome and merged using FluoView software (Olympus, Tokyo, Japan) as previously described 24 .…”

“…Because of its physiological role in reducing hepcidin levels, it stands to reason that TMPRSS6 has become an attractive therapeutic target for diseases characterized by iron overload, such as hereditary haemochromatosis (OMIM #235200) and beta-thalassemia (OMIM #613985) 16 – 18 . Therefore, to further understand the role of TMPRSS6 in iron homeostasis, cellular models including primary hepatocytes and liver-derived hepatocyte cell lines, such as hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), have been widely used because they possess both the protein and signalling machinery controlling hepcidin expression 3 , 19 – 24 .…”

A Short Peptide Inhibitor as an Activity-Based Probe for Matriptase-2

“…Such a probe might also circumvent the lack of specific antibodies against MT-2. Even though biotin-RQRR-CMK is not selective for MT-2 over matriptase, it can be considered a viable tool for the screening and visualization of active MT-2, since the expression patterns of both enzymes are different [ 3 , 39 ].…”

“…This group includes enzymes such as thrombin and trypsin, which are critical in the process of blood coagulation and digestion. Our enzyme of interest, matriptase-2 (MT-2), a member of the type II transmembrane serine proteases (TTSPs) [ 1 , 2 ], is mainly expressed in the liver [ 3 ] and considered as a key regulator of iron homeostasis [ 4 , 5 ]. By cleaving membrane-bound hemojuvelin (HJV), the co-receptor of the bone morphogenic protein (BMP) receptor, MT-2 decreases the activation of the SMAD signaling cascade and, in consequence, the expression of the hepcidin gene Hamp [ 6 , 7 ].…”